Prokaryotic repressor proteins regulate gene expression through sequence-specific recognition of DNA. The aim of this grant application is to investigate the biophysical origins of specificity through heteronuclear NMR studies of the Lambda repressor-operator complex. An interdisciplinary approach, based upon two-dimensional NMR techniques and modern genetic methods, will enable structure-function relationships to be studied. In addition this proposal develops NMR techniques that will be of general utility in the study of macromolecules of biological interest. High-resolution 1H and 13C-NMR spectroscopy will be used to study the conformation and dynamic of Lambda repressor and its interaction with Lambda operator. For this purpose, we will selectively label repressor with 13C-enriched amino acids by biosynthetic incorporation. These labels provide intrinsic non-perturbing probes for protein structure and dynamics which can be monitored in the protein-DNA complex. Two-dimensional 1H-13C-NMR experiments will be used to enhance resolution and allow proton resonances to be monitored simultaneously. Selective labeling simplifies the 13C-NMR spectrum, facilitates resonance assignment and overcomes the sensitivity limitations of natural abundance 13C-NMR studies. Mutant repressors bearing single-amino acid substitutions will be studied to probe structure-function relationships and to make regorous assignment of NMR-resonances. Mutant operator sites, which provide specific perturbations of the complex, will also be examined. By means of these probes, the following will be investigated: (a) the helix-turn-helix motif as a general framework of sequence-specific recognition, (b) the N-terminal arm, which is thought to wrap around the DNA in the repressor-operator complex, and (c) the structure and dynamics of operator DNA. The experimental information obtained will be used to test and refine models of the repressor-operator complex. It is expected that this interdisciplinary strategy will lead to a deeper understanding of the remarkable specificity inherent in operator recognition.